Spin-labeled hemoglobin derivatives in solution and in single crystals.

Abstract

The paramagnetic resonance of a spin-label attached to a protein in solution, or in a single crystal, depends on static as well as on the dynamic features of its molecular environment.' This conformation-dependent paramagnetic resonance offers the possibility of relating structural properties of proteins in single crystals to those in solutions. The present paper is a brief report of a study of this type.Here we compare the paramagnetic resonance spectra of spin-labeled horse hemoglobin in solution, and in single crystals, for the carbonmonoxyand acid met (ferric) derivatives. The present work was stimulated by an earlier, puzzling observation that the paramagnetic resonance spectra of spin-labeled horse oxy-and acid methemoglobin in solution are significantly different,2 whereas Perutz and co-workers have shown that these two molecules must have very similar secondary and tertiary structures, and identical quaternary structures in the crystalline state. 3 4 Materials and Methods.-The spin-label used in the present work is N-This label, analogous to the five-membered nitroxide ring iodoacetamide label described previously,2 was prepared as follows: Commercially available 2,2,6,6-tetramethyl-4-aminopiperidine (I) was acetylated at O0 in acetic anhydride.-The resulting 2,2,6,6-tetramethyl-4-acetamidopiperidine (II) was treated with hydrogen peroxide in the standard manner6' 7 to give the corresponding nitroxide (III) in 65% yield (based on the starting material I) after recrystallization from toluene, mp 139-141' (uncorrected). Hydrolysis of III with refluxing 10% sodium hydroxide gave a 72% yield of hygroscopic 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl (IV), which was purified by distillation, bp 61-63°(1 mm). The product crystallized in the receiver. Coupling of (IV) with iodoacetic acid that was freshly recrystallized from benzene-hexane was accomplished with pure dicyclohexylcarbodiimide in ethyl acetate.8 Chromatography on neutral alumina followed by recrystallization from toluene-hexane gave label V in 60% yield, mp 118.5-120.5o (uncorrected).Horse hemoglobin was prepared by the method of Benesch and Benesch.9 The hemoglobin was labeled with the iodoacetamide V, using procedures similar to those described previously.2 The crystals were prepared with a recipe kindly given to us by Dr. M. F. Perutz:4 Hemoglobin crystals were obtained from a series of vials, each containing 2 ml of 2% labeled carbon monoxide hemoglobin (HbCO), or methemoglobin (met Hb), and between 2 and 3 ml of solution "A." Solution "A" contained 2 vol of 4 M (NH4)2SO4 and 1 vol of 2 M (NH4)2HP04. After crystals were formed they were "cured" by addition of ca. 0.3 ml of a 1.8 M NH4H2PO4 solution to 4-5 ml of the suspension medium. This brought the final crystal pH to 6.9 or 7.0. No significant changes in the resonance spectra of the single crystals were detected in the range pH 6.8-7.2. In the present study we used crystals of HbCO rather than oxyhemoglobin (HbO2) in order to reduce the likelihood of contamination by met Hb. 77"Downloaded...

Conformational Equilibria in Spin-Labeled Hemoglobin

“…These results demonstrate several things: (i) The controls of semiconservative DNA replication do not extend to repair synthesis; (ii) differentiated skeletal muscle cells can phosphorylate and incorporate thymidine into acid-insoluble material within each muscle nucleu; (iii) a polymerizing enzyme must be present in sufficient amount for this to occur; and (iv) this synthetic 6. Experiments to be reported elsewhere demonstrate that thymidine dimers are formed in this system when DNA is exposed to ultravio-19 MARCH 1971 let light and that tritiated thymidine is incorporated into high molecular weight material which is resistant to alkaline hydrolysis and which moves with DNA in cesium chloride gradients.…”

“…Procedures for preparing spin labels 1 and 2 and for isolating and spinlabeling hemoglobin have been described (2,6,7). Conversion to deoxyhemoglobin was determined by the spectrophotometric method of Benesch (8).…”

Electron paramagnetic resonance as a unique tool for skin and hair research

“…In particular, it covers synthesis of new spin traps and spin labels including spin‐labelled drugs and cosmeceutics, improvement of solid oximetric spin probes injected intradermally or implanted subcutaneously (128,130,131,199), development of EPR in vivo measurement of thiols (81), extensive exploration of skin melanin leading to in situ measurement of melanin contents (54) in pigmented naevi, melanoma tumors, sun‐tanned skin, or to follow the process of adaptation of transplanted melanocytes in piebald or vitiligo skin (200), and to explore the pigment as a natural spin probe or a contrast agent in EPRI (54). A combination of spin labelling (107,122,123) and molecular biology techniques (site‐directed spin labelling, SDSL) (201) should deliver proteins of dermatological importance with spin label groups substituted in any necessary site of a molecule (4, 202).…”

“…melanoma tumors, sun-tanned skin, or to follow the process of adaptation of transplanted melanocytes in piebald or vitiligo skin (200), and to explore the pigment as a natural spin probe or a contrast agent in EPRI (54). A combination of spin labelling (107,122,123) and molecular biology techniques (site-directed spin labelling, SDSL) (201) should deliver proteins of dermatological importance with spin label groups substituted in any necessary site of a molecule (4,202).…”

Electron Paramagnetic Resonance Studies of Spin-Labeled Hemoglobins and Their Implications to the Nature of Cooperative Oxygen Binding to Hemoglobin

“…21 The procedure for introducing the spin label to hemoglobin was the same as that described by Ogawa et al281 In our studies, we have used HbCO instead of HbO2. This is particularly advantageous because Hb Zurich and Hb Chesapeake are very sensitive to autooxidation.…”